1. Field of the Invention
The present invention relates to a method for controlling the molecular weight of a polyhydroxyalkanoate (PHA), a kind of polyester. More specifically, the present invention relates to a method for controlling the molecular weight of the PHA employing a microorganism which is capable of producing and accumulating the PHA in the cell.
2. Related Background Art
Many microorganisms have been reported to produce and accumulate poly-3-hydroxybutyric acid (PHB) or other PHA in the cells (xe2x80x9cSeibunkaisei Purasutikku Handobukku (Biodegradable Plastics Handbook)xe2x80x9d, Biodegradable plastics Research Group, NTS K. K., pp.178-197 (1995)). These polymers are useful as various articles molded by melt processing or other processing similarly as conventional polymers. Moreover, these polymers, which are biodegradable, are completely degraded by a microorganism in the nature, not causing pollution in natural environment advantageously, differently from conventional synthetic high polymers. Furthermore, these diodegradable polymers are highly adaptable to a living body and are promising also for a medical soft material and other uses.
Such PHAs produced by a microorganism are known to have various compositions and structures depending on the kind of microorganism, culture medium composition, cultivation conditions, and other factors in the production. The control of the composition and structure of PHA has been studied for improvement of the physical properties of PHA.
The PHAs produced by microorganisms are roughly classified into two groups according to the biosynthesis mechanism. One group of PHAs are short-chain-length PHAs (hereinafter referred to as xe2x80x9cscl-PHA(s)xe2x80x9d) typified by polyhydroxybutyric acid (PHB), polyhydroxyvaleric acid (PHV), and copolymers thereof: the other group of PHAs are medium-chain-length PHAs (hereinafter referred to as xe2x80x9cmcl-PHA(s)xe2x80x9d) having medium-chain-length 3-hydroxyalkanoic acid of about 6-14 carbons as the units.
The former, scl-PHA, is formed from sugars such as glucose and gluconic acid, or acetyl-CoA which is an in-vivo metabolism product of organic acids such as lactic acid, pyruvic acid, and malic acid as the starting material by enzymatic dimerization and reduction into a polymer.
The latter, mcl-PHA, is formed enzymatically from an alkanoic acid as the starting material by CoA addition, dehydrogenation, and water addition through the xcex2-oxidation pathway, a fatty acid degradation system, into a polymer.
As mentioned above, the respective groups of PHAs are synthesized through different biosynthesis pathways by action of different enzymes in vivo according to the results of detailed investigation.
Of the microorganisms producing the latter, mcl-PHAs, some microorganisms are known to produce PHAs having various functional groups and residues.
Among them, production of the PHAs having an aromatic ring in the unit is actively investigated in recent years.
Makromol.Chem., 191, 1957-1965 (1990) and Macromolecules, 24, 5256-5260 (1991) describe that Pseudomonas oleovorans produces a PHA containing 3-hydroxy-5-phenylvaleric acid as the unit from 5-phenylvaleric acid as the substrate.
Macromolecules, 29, 1762-1766 (1996) describes that Pseudomonas oleovorans produces a PHA containing 3-hydroxy-5-(4xe2x80x2-tolyl)valeric acid as the unit from 5-(4xe2x80x2-tolyl)valeric acid as the substrate.
Macromolecules, 32, 2889-2895 (1999) describes that Pseudomonas oleovorans produces a PHA containing 3-hydroxy-5-(2xe2x80x2,4xe2x80x2-dinitrophenyl)valeric acid and 3-hydroxy-5-(4xe2x80x2-nitrophenyl)valeric acid as the units from 5-(2xe2x80x2,4xe2x80x2-dinitrophenyl)valeric acid as the substrate.
Macromol.Chem.Phys., 195, 1665-1672 (1994) describes that Pseudomonas oleovorans produces a PHA copolymer of 3-hydroxy-5-phenoxyvaleric acid and 3-hydroxy-9-phenoxynonanoic acid from 11-phenoxyundecanoic acid as the substrate.
Japanese Patent Publication No. 2989175 discloses homopolymers having a 3-hydroxy-5-(monofluorophenoxy)pentanoate (3H5(MHP)P) unit or a 3-hydroxy-5-(difluorophenoxy)pentanoate (3H5(DHP)P) unit, and copolymers having at least the 3H5(MFP)P unit or 3H5(DFP)P unit synthesized by Psudomonas putida (Pseudomanas Genus); and a process for synthesis of the above polymers. Thereby, a polymer having a mono- or di-fluorine-substituted phenoxy group at side chain ends can be synthesized by assimilation of a substituted long-chain fatty acid, the polymer having stereoregularity and water repellency with retention of the high melting point and high processability.
Cyano- or nitro-substituted polymers are investigated besides the above fluorine-substituted polymers.
Can.J.Microbiol., 41, 32-43 (1995), and Polymer International, 39, 205-213 (1996) describe production of PHAs containing 3-hydroxy-p-cyanophenoxyhexanoic acid or 3-hydroxy-p-nitrophenoxyhexanoic acid as the monomer unit from octanoic acid and p-cyanophenoxyhexanoic acid or p-nitrophenoxyhexanoic acid as the substrate by employing Pseudomonas oleovorans ATCC29347 strain and Pseudomonas putida KT2442 strain.
Macromolecules, 32, 8315-8318 (1999) and Polymer Preprints, Japan, 49(5), 1034 (2000) describe capability of Pseudomonas putida 27N01 strain to produce PHA copolymers containing 3-hydroxy-5-thiophenoxyvaleric acid and 3-hydroxy-7-thiophenoxyheptanoic acid from 11-thiophenoxyvaleric acid as the substrate.
For practical application of the PHAs, control of the molecular weight is attempted to broaden the application field thereof.
U.S. Pat. No. 6,156,852 discloses the decrease of the number-average molecular weight in biosynthesis of PHB by employing Ralstonia eutropha, Ralstonia latus, and Comamonas testosteroni as the producing microorganism strain by addition of a diol such as ethylene glycol, neopentyl glycol, propylene glycol, butanediol, hexanediol, and octanediol; butanetriol, polypropylene glycol, glycerol, hydroquinone, benzene-dimethanol, pentaerithritol, and derivatives thereof; or a sugar alcohol such as sorbitol and mannitol to the culture medium. These items are described in detail as chemical reports in Biotechnology and Bioengineering, 62, 106-113 (1999), and International Journal of Biological Macromolecules, 25, 43-53 (1999).
These techniques have merits of controlling the molecular weight in the PHA biosynthesis process without using a chemical substance such as an acid or a base. The PHAs having a functional group such as the phenyl group described above are also required to be controlled in the molecular weight for broadening the practical application field. However, no technique therefore has been developed yet.
The present invention provide a method for controlling the molecular weight of a polyhydroxyalkanoate having units of a residue containing a phenyl-, thienyl-, or cyclohexyl-structure in the side chain of the molecule.
After comprehensive study to solve the above problems, the inventors of the present invention achieved the invention described below.
The present invention provides a method for controlling the molecular weight of a polyhydroxyalkanoate containing at least one of 3-hydroxy-xcfx89-substituted alkanoic acid units represented by Chemical Formula (1): 
(in the above formula, m is an integer selected from the numerical range shown with the Chemical Formula;
R1 is a residue having a ring structure of any one selected from the group consisting of a phenyl structure and a thienyl structure; and in the presence of plural units, m and R1 are selected independently for the respective units), and 3-hydroxy-xcfx89-cyclohexylalkanoic acid units represented by Chemical Formula (2): 
(in the above formula, R2 denotes a substituent on the cyclohexyl group selected from the group consisting of H atom, CN, NO2, halogen atom, CH3, C2H5, C3H7, CF3, C2F5 and C3F7; k is an integer selected from the numerical range shown with the Chemical Formula;
and in the presence of plural units, k and R2 are selected independently for the respective units), wherein a microorganism is cultivated, in the presence of a hydroxyl group-containing compound, which is capable of producing the polyhydroxyalkanoate containing at least one of the units represented by Chemical Formula (1) or (2) from an xcfx89-substituted alkanoic acid represented by Chemical Formula (3): 
(in the above formula, q is an integer selected from the numerical range shown with the Chemical Formula;
R3 is a residue having a ring structure of any one selected from the group consisting of a phenyl structure and a thienyl structure; and in the presence of plural units, q and R3 are selected independently for the respective units), or xcfx89-cyclohexylalkanoic acid represented by Chemical Formula (4): 
(in the above formula, R4 denotes a substituent on the cyclohexyl group selected from the group consisting of H atom, CN, NO2, halogen atom, CH3, C2H5, C3H7, CF3, C2F5 and C3F7; and r is an integer selected from the numerical range shown with the Chemical Formula; and in the presence of plural units, R4 and r are selected independently for the respective units).
Here, R1, and R3 in Chemical Formula (1) or (3), namely the residue having the phenyl structure or the thienyl structure includes specifically the groups represented by Chemical Formulas (5) to (15):
a substituted or unsubstituted phenyl group represented by General Formula (5): 
(in the above formula, R5 denotes a substituent on the aromatic ring selected from the group consisting of H atom, halogen atom, CN, NO2, CH3, C2H5, C3H7, vinyl, COOR51 (for R1 only; R51 is a substituent selected from the group consisting of H atom, Na atom and K atom), CF3, C2F5 and C3F7; and in the presence of plural units, the above definition is applied independently of the respective units);
a substituted or unsubstituted phenoxy group represented by General Formula (6): 
(in the above formula, R6 denotes a substituent on the aromatic ring selected from the group consisting of H atom, halogen atom, CN, NO2, CH3, C2H5, C3H7, SCH3, CF3, C2F5 and C3F7; and in the presence of plural units, the above definition is applied independently of the respective units);
a substituted or unsubstituted benzoyl group represented by General Formula (7): 
(in the above formula, R7 denotes a substituent on the aromatic ring selected from the group consisting of H atom, halogen atom, CN, NO2, CH3, C2H5, C3H7, CF3, C2F5 and C3F7; and in the presence of plural units, the above definition is applied independently of the respective units);
a substituted or unsubstituted phenylsulfanyl group represented by General Formula (8): 
(in the above formula, R8 denotes a substituent on the aromatic ring selected from the group consisting of H atom, halogen atom, CN, NO2, COOR81, SO2R82 (R81 is a substituent selected from the group consisting of H, Na, K, CH3 and C2H5; R82 is a substituent selected from the group consisting of OH, ONa, OK, halogen atom, OCH3 and OC2H5), CH3, C2H5, C3H7, (CH3)2xe2x80x94CH and (CH3)3xe2x80x94C; and in the presence of plural units, the above definition is applied independently of the respective units);
a substituted or unsubstituted (phenylmethyl)sulfanyl group represented by General Formula (9): 
(in the above formula, R9 denotes a substituent on the aromatic ring selected from the group consisting of H atom, halogen atom, CN, NO2, COOR91, SO2R92 (R91 is a substituent selected from the group consisting of H, Na, K, CH3 and C2H5; R92 is a substituent selected from the group consisting of OH, ONa, OK, halogen atom, OCH3 and OC2H5), CH3, C2H5, C3H7, (CH3)2xe2x80x94CH and (CH3)3xe2x80x94C; and in the presence of plural units, the above definition is applied independently of the respective units);
a 2-thienyl group represented by Chemical Formula (10): 
a 2-thienylsulfanyl group represented by Chemical Formula (11): 
a 2-thienylcarbonyl group represented by Chemical Formula (12): 
a substituted or unsubstituted phenylsulfinyl group represented by General Formula (13) (for R1 only) 
(in the above formula, R13 denotes a substituent on the aromatic ring selected from the group consisting of H atom, halogen atom, CN, NO2, COOR131, SO2R132 (R131 is a substituent selected from the group consisting of H, Na, K, CH3 and C2H5; R132 is a substituent selected from the group consisting of OH, ONa, OK, halogen atom, OCH3 and OC2H5), CH3, C2H5, C3H7, (CH3)2xe2x80x94CH and (CH3)3xe2x80x94C; and in the presence of plural units, the above definition is applied independently of the respective units);
a substituted or unsubstituted phenylsulfonyl group represented by General Formula (14) (for R1 only) 
(in the above formula, R14 denotes a substituent on the aromatic ring selected from the group consisting of H atom, halogen atom, CN, NO2, COOR141, SO2R142 (R141 representing H, Na, K, CH3 and C2H5; R142 is a substituent selected from the group consisting of OH, ONa, OK, halogen atom, OCH3 and OC2H5), CH3, C2H5, C3H7, (CH3)2xe2x80x94CH and (CH3)3xe2x80x94C; and in the presence of plural units, the above definition is applied independently of the respective units); and
a (phenylmethyl)oxy group represented by Chemical Formula (15): 
The hydroxyl group-containing compound is at least the one selected from the group consisting of alcohols, diols, triols, alkylene glycols, polyethylene glycols, polyethylene oxides, alkylene glycol monoesters, polyethylene glycol monoesters, and polyethylene oxide monoesters.
More specifically, the alcohols, diols, and triols may include linear and branched alcohols, diols, and triols of 3-14 carbons.
The alkylene glycols and alkylene glycol monoesters may be compounds of 2-10 carbons having a linear or branched structure.
The polyethylene glycols, polyethylene oxides, polyethylene glycol monoesters, and polyethylene oxide monoesters may have a number-average molecular weight ranging from 100 to 20000.
The hydroxyl group-containing compound is preferably added to the culture medium for cultivation of the microorganism at a concentration preferably from 0.01 to 10%(w/v), more preferably from 0.02 to 5%(w/v). The compound may be added in one batch in the initial stage of the cultivation, or portionwise during the cultivation period.
The microorganism employed in the present invention is the one which has, as described above, capability of producing a polyhydroxyalkanoate containing at least one of 3-hydroxy-xcfx89-substituted alkanoic acid units represented by Chemical Formula (1), and a 3-hydroxy-xcfx89-cyclohexylalkanoic acid unit represented by Chemical Formula (2), from an xcfx89-substituted alkanoic acid, as the source material, represented by Chemical Formula (3), or an xcfx89-cyclohexylalkanoic acid represented by Chemical Formula (4).
The polyhydroxyalkanoate of the present invention contains at least one of 3-hydroxy-xcfx89-substituted-alkanoic acid units represented by Chemical Formula (16): 
(in the above formula, m is an integer selected from the numerical range shown with the Chemical Formula; R1 is defined in above and preferably a residue selected from the group consisting of Chemical formulae (5) to (15); in the presence of plural units, m and R1 are selected independently for the respective units; and R16 is a group derived from a chemical species selected from the group consisting of alcohols, diols, triols, alkylene glycols, polyethylene glycols, polyethylene oxides, alkylene glycol monoesters, polyethylene glycol monoesters, and polyethylene oxide monoesters); and
3-hydroxy-xcfx89-cyclohexylalkanoic acid units represented by General formula (17): 
(in the above formula, R2 denotes a substituent on the cyclohexyl group selected from the group consisting of H atom, CN, NO2, halogen atom, CH3, C2H5, C3H7, CF3, C2F5 and C3F7; k is an integer selected from the numerical range shown with the Chemical Formula; in the presence of plural units, k and R2 are selected independently for the respective units; and
R17 is a group derived from a chemical species selected from the group consisting of alcohols, diols, triols, alkylene glycols, polyethylene glycols, polyethylene oxides, alkylene glycol monoesters, polyethylene glycol monoesters, and polyethylene oxide monoesters).
The use of the aforementioned microorganism is the essential constitutional requirement of the present invention. Specifically, the microorganisms employed in U.S. Pat. No. 6,156,852, Biotechnology and Bioengineering, 62, 106-113 (1999), and International Journal of Biological Macromolecules, 25, 43-53 (1999) do not have capability of producing the polyhydroxyalkanoate containing one or more of the units represented by Chemical Formula (1) or (2) starting from the compound represented by Chemical Formula (3) or (4)
The microorganisms shown in the above Japanese Patent Publication and the technical literature are reported to produce usually homopolymers and copolymers of poly 3-hydroxybutyric acid (hereinafter referred to as xe2x80x9cPHBxe2x80x9d), or poly 3-hydroxyvaleric acid (hereinafter referred to as xe2x80x9cPHVxe2x80x9d). Typically, the biosynthesis pathway for the PHB is summarized as below:
(1) Acetyl-CoAxe2x86x92Acetoacetyl-CoA
(2) Acetoacetyl-CoAxe2x86x923-hydroxybutyryl-CoA
(3) 3-Hydroxybutyryl-CoAxe2x86x92poly 3-hydroxybutric acid.
On the other hand, the microorganism employed in the present invention biosynthesizes a polyhydroxyalkanoate containing one or more of the units represented by Chemical Formula (1) and (2) by taking the compound of Chemical Formula (3) or (4) into the fatty acid degradation pathway called xe2x80x9cxcex2-oxidation pathwayxe2x80x9d by conversion as shown below:
 less than 1 greater than  Compound (2)xe2x86x92Acyl-CoA
 less than 2 greater than  Acyl-CoAxe2x86x92Enoyl-CoA
 less than 3 greater than  Enoyl-CoAxe2x86x923-Hydorxyacyl-CoA
 less than 4 greater than  3-Hydroxyacyl-CoAxe2x86x92Polyhydroxyalkanoate of Chemical Formula (1).
The enzyme participating directly in the above step (3) is a PHB synthase or a short-chain-length PHA synthase, whereas the enzyme employed in the step  less than 4 greater than  of the present invention is a PHA synthase or a medium-chain-length PHA synthase. The both enzymes are different from each other in the substrate specificity. This is shown in detail in review papers such as FEMS microbiology Reviews, 103, 217-230 (1992), and Journal of Biotechnology, 65, 127-161 (1998).
In other words, the microorganism employed in the present invention is completely different from the microorganisms employed in the aforementioned U.S. Pat. No. 6,156,852; Biotechnology and Bioengineering, 62 106-113 (1999); and International Journal of Biological Micromolecules, 25, 43-53 (1999) cited above under the heading of Related Background Art in this Patent Specification.
The microorganism employed in the method of the present invention is not limited, provided that the microorganism has capability of producing the polyhydroxyalkanoate containing at least one of the units represented by Chemical Formula (1) or (2) in the molecule from a source compound represented by Chemical Formula (3) or (4). Among them, particularly preferred are microorganisms of Pseudomonas genus, including specifically Pseudomonas cichorii, Pseudomonas putida, Pseudomonas fluorescence, Pseudomonas oleovorans, Pseudomonas aeruginosa, Pseudomonas stutzeri, Peudomonas jessenii, and so forth. More specifically, particularly preferred are Pseudomonas cichori YN2 (FERM BP-7375), Pseudomonas cichorii H45 (FREM BP-7374), Pseudomonas jessenii P161 (FERM BP-7376), and Pseudomonas putida P91 (FREM BP-7373). These four microorganisms have been deposited to International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (formerly, National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology), and are described in Japanese Patent Application Laid-Open No. 2001-371863.
The microorganism cultivating conditions in the present invention are described below.
Necessary substrate and nutrients are added as explained below to an inorganic culture medium basically constituted of a phosphate buffer, and an ammonium or nitrate salt.
As the substrate for production of the intended polyhydroxyalkanoate, the culture medium contains the compound of Chemical Formula (2) preferably at a content ranging from 0.01 to 1%(w/v), more preferably from 0.02 to 0.2%(w/v).
Usually the culture medium contains preferably the coexisting substrate below as the carbon source and nitrogen source for the microorganism growth, and energy source for the polyalkanoate production at a concentration ranging preferably from 0.1 to 5%(w/v), more preferably from 0.2 to 2%.
Coexisting Substrate:
Natural Culture Component: yeast extract, meat extract, malt extract, kazaminic acid, casein hydrolyzate, polypeptone, trypton, peptone, etc.;
Sugars: aldoses such as glyceroaldehyde, erythrose, arabinose, xylose, glucose, galactose, mannose, and fructose; alditol such as glycerol, erythritol and xylitol; aldonic acids such as gluconic acid; uronic acids such as glucuronic acid and galacturonic acid; disaccharides such as maltose, sucrose and lactose;
Organic acids and salts thereof: pyruvic acid, malic acid, lactic acid, citric acid, succinic acid, oxaloacetic acid, isocitric acid, ketoglutaric acid and fumaric acid, and salts thereof;
Amino acids: glutamic acid, aspartic acid, and salts thereof, etc.;
Alkanoic acids: linear or branched alkanoic acids of 4-12 carbons, etc.
In the present invention, any inorganic culture medium is useful which contains a phosphate salt and a nitrogen source such as an ammonium or nitrate salt. The productivity of PHA can be improved by controlling the concentration of the nitrogen source.
The cultivating temperature is controlled to be suitable for the growth of the aforementioned microorganism strain, ranging preferably from 15xc2x0 C. to 37xc2x0 C., more preferably from 20xc2x0 C. to 30xc2x0 C.
Any cultivation method may be employed which allows growth of the microorganism to produce PHA, including liquid cultivation and solid cultivation production. The cultivation may be conducted by any kind of cultivation process such as batch cultivation, fed-batch cultivation, semi-continuous cultivation and continuous cultivation. The liquid cultivation method includes a flask-shaking method for oxygen supply, and stirring aeration with a jar fermentor for oxygen supply.
In another method for producing and accumulating the PHA in the microorganism, the microorganism is allowed to grow sufficiently, then the microorganism mass is transferred to a separate culture medium containing a limited amount of a nitrogen source like ammonium chloride, and the cultivation is continued with addition of the compound for the substrate of the intended units to improve the productivity possibly.
The objective PHA can be isolated from the microorganism cells after cultivation as described above in a conventional process in the present invention. For example, the most simplest process is extraction with an organic solvent like chloroform, dichloromethane, and acetone. Another organic solvent such as dioxane, tetrahydrofuran and acetonitrile can be useful. In the environment in which use of the organic solvent is not suitable, PHA can be recovered by physically crushing the microorganism cells to remove the cell component other than PHA by any of the methods: treatment with a surfactant like SDS; treatment with an enzyme like lysozyme; by treatment with a chemical such as hypochlorite salts, ammonia and EDTA; and physical crushing of the microorganism cells by ultrasonic crushing, homogenizer crushing, pressure crushing, bead-impact crushing, milling, grinding, and freeze-melting.
Incidentally, in the present invention, the methods are not limited to the above for cultivation of the microorganism, production and accumulation of PHA in the microorganism cells, and the recovery of PHA by removal of the microorganism cell components other than PHA.
As shown in the following working examples, the method of the resent invention enables control of the molecular weight of polyhydroxyalkanoate which contains a unit having a residue containing a phenyl-, thienyl-, or cyclohexyl-structure in the side chain of the molecule.
The composition of the inorganic salt culture medium (M9 culture medium) which was used in a method of the present invention is shown below.
For more effective cell growth and PHS production, the minor component solutions should be added to the culture medium in an amount of about 0.3%(v/v) as shown below.
Nitrilotriacetic Acid:1.5; MgSO4:3.0; MnSO4:0.5; NaCl:1.0; FeSO4:0.1; CaCl2:0.1; CoCl2:0.1; ZnSO4:0.1; CuSO4:0.1; AlK(SO4)2:0.1; H3BO3:0.1; Na2MoO4:0.1; NiCl2:0.1 (g/L)